. 2022 Mar 4;375(6584):eabk2432.

doi: 10.1126/science.abk2432. Epub 2022 Mar 4.

Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly

Hongjie Li^#^{1

2

3}, Jasper Janssens^#^{4

5}, Maxime De Waegeneer^{4

5}, Sai Saroja Kolluru^{6

7

8}, Kristofer Davie⁴, Vincent Gardeux^{9

10}, Wouter Saelens^{9

10}, Fabrice P A David^{9

10

11}, Maria Brbić^{8

12}, Katina Spanier^{4

5}, Jure Leskovec^{8

12}, Colleen N McLaughlin¹, Qijing Xie¹, Robert C Jones^{6

7

8}, Katja Brueckner¹³, Jiwon Shim¹⁴, Sudhir Gopal Tattikota^{15

16}, Frank Schnorrer¹⁷, Katja Rust^{18

19}, Todd G Nystul¹⁹, Zita Carvalho-Santos²⁰, Carlos Ribeiro²⁰, Soumitra Pal²¹, Sharvani Mahadevaraju²², Teresa M Przytycka²¹, Aaron M Allen²³, Stephen F Goodwin²³, Cameron W Berry²⁴, Margaret T Fuller²⁴, Helen White-Cooper²⁵, Erika L Matunis²⁶, Stephen DiNardo^{27

28}, Anthony Galenza²⁹, Lucy Erin O'Brien²⁹, Julian A T Dow³⁰; FCA Consortium§; Heinrich Jasper³¹, Brian Oliver²², Norbert Perrimon^{15

16}, Bart Deplancke^{9

10}, Stephen R Quake^{6

7

8}, Liqun Luo¹, Stein Aerts^{4

5}, Devika Agarwal³², Yasir Ahmed-Braimah³³, Michelle Arbeitman³⁴, Majd M Ariss³⁵, Jordan Augsburger¹³, Kumar Ayush¹², Catherine C Baker³⁶, Torsten Banisch³⁷, Katja Birker³⁸, Rolf Bodmer³⁸, Benjamin Bolival³⁶, Susanna E Brantley³⁶, Julie A Brill^{39

40}, Nora C Brown⁴¹, Norene A Buehner⁴¹, Xiaoyu Tracy Cai³¹, Rita Cardoso-Figueiredo²⁰, Fernando Casares⁴², Amy Chang⁴³, Thomas R Clandinin⁴⁴, Sheela Crasta^{6

7

8}, Claude Desplan⁴⁵, Angela M Detweiler⁸, Darshan B Dhakan²⁰, Erika Donà⁴⁶, Stefanie Engert⁴³, Swann Floc'hlay^{4

5}, Nancy George⁴⁷, Amanda J González-Segarra⁴³, Andrew K Groves^{3

48}, Samantha Gumbin²⁹, Yanmeng Guo^{49

50}, Devon E Harris³⁶, Yael Heifetz⁵¹, Stephen L Holtz⁵², Felix Horns⁵³, Bruno Hudry⁵⁴, Ruei-Jiun Hung¹⁵, Yuh Nung Jan^{49

50}, Jacob S Jaszczak^{49

50}, Gregory S X E Jefferis⁴⁶, Jim Karkanias⁸, Timothy L Karr⁵⁵, Nadja Sandra Katheder³¹, James Kezos³⁸, Anna A Kim^{29

56

57}, Seung K Kim^{24

58}, Lutz Kockel²⁴, Nikolaos Konstantinides⁵⁹, Thomas B Kornberg⁶⁰, Henry M Krause⁶¹, Andrew Thomas Labott²⁹, Meghan Laturney⁴³, Ruth Lehmann⁶², Sarah Leinwand⁴³, Jiefu Li¹, Joshua Shing Shun Li¹⁵, Kai Li^{49

50}, Ke Li^{49

50}, Liying Li^{49

50}, Tun Li^{49

50}, Maria Litovchenko^{9

10}, Han-Hsuan Liu^{49

50}, Yifang Liu¹⁵, Tzu-Chiao Lu², Jonathan Manning⁴⁷, Anjeli Mase¹³, Mikaela Matera-Vatnick⁴¹, Neuza Reis Matias³⁶, Caitlin E McDonough-Goldstein^{63

64}, Aaron McGeever⁸, Alex D McLachlan⁶⁵, Paola Moreno-Roman²⁹, Norma Neff⁸, Megan Neville²³, Sang Ngo²⁹, Tanja Nielsen³⁸, Caitlin E O'Brien^{49

50}, David Osumi-Sutherland⁶⁶, Mehmet Neset Özel⁶⁷, Irene Papatheodorou⁴⁷, Maja Petkovic^{49

50}, Clare Pilgrim⁶⁸, Angela Oliveira Pisco⁸, Carolina Reisenman⁴³, Erin Nicole Sanders²⁹, Gilberto Dos Santos⁶⁹, Kristin Scott⁴³, Aparna Sherlekar²⁹, Philip Shiu⁴³, David Sims³², Rene V Sit⁸, Maija Slaidina⁷⁰, Harold E Smith⁷¹, Gabriella Sterne⁴³, Yu-Han Su²⁹, Daniel Sutton⁷², Marco Tamayo³⁸, Michelle Tan⁸, Ibrahim Tastekin²⁰, Christoph Treiber⁷³, David Vacek¹, Georg Vogler³⁸, Scott Waddell⁷³, Wanpeng Wang⁶⁰, Rachel I Wilson⁵², Mariana F Wolfner⁴¹, Yiu-Cheung E Wong³⁶, Anthony Xie¹, Jun Xu¹⁵, Shinya Yamamoto^{3

74}, Jia Yan⁸, Zepeng Yao⁴³, Kazuki Yoda²⁹, Ruijun Zhu^{49

50}, Robert P Zinzen⁷⁵

Affiliations

¹ Howard Hughes Medical Institute, Department of Biology, Stanford University, Stanford, CA 94305, USA.
² Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA.
³ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
⁴ VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, Leuven 3000, Belgium.
⁵ Laboratory of Computational Biology, Department of Human Genetics, KU Leuven, Leuven 3000, Belgium.
⁶ Department of Bioengineering, Stanford University, Stanford, CA, USA.
⁷ Department of Applied Physics, Stanford University, Stanford, CA, USA.
⁸ Chan Zuckerberg Biohub, San Francisco CA, USA.
⁹ Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
¹⁰ Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.
¹¹ Bioinformatics Competence Center, EPFL, Switzerland.
¹² Department of Computer Science, Stanford University, Stanford, CA 94305, USA.
¹³ Department of Cell and Tissue Biology, University of California, San Francisco, CA 94143, USA.
¹⁴ Department of Life Science, College of Natural Science, Hanyang University, 04763 Seoul, Republic of Korea.
¹⁵ Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
¹⁶ Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.
¹⁷ Aix-Marseille University, CNRS, IBDM (UMR 7288), Turing Centre for Living Systems, 13009 Marseille, France.
¹⁸ Institute of Physiology and Pathophysiology, Department of Molecular Cell Physiology, Philipps-University, Marburg, Germany.
¹⁹ Department of Anatomy, University of California, San Francisco, CA 94143, USA.
²⁰ Behavior and Metabolism Laboratory, Champalimaud Research, Champalimaud Centre for the Unknown, Lisbon, Portugal.
²¹ National Center of Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
²² Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Kidney and Digestive Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
²³ Centre for Neural Circuits and Behaviour, University of Oxford, Oxford OX1 3SR, UK.
²⁴ Department of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
²⁵ Molecular Biosciences Division, Cardiff University, Cardiff CF10 3AX, UK.
²⁶ Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
²⁷ Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA 19104, USA.
²⁸ The Penn Institute for Regenerative Medicine, Philadelphia, PA 19104, USA.
²⁹ Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
³⁰ Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
³¹ Immunology Discovery, Genentech, Inc., South San Francisco, CA 94080, USA.
³² MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
³³ Department of Biology, Syracuse University, Syracuse, NY 13244 USA.
³⁴ Biomedical Sciences Department, Florida State University, Tallahassee, FL, USA.
³⁵ Department of Genetics, Harvard Medical School, Boston, MA, USA.
³⁶ Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
³⁷ Skirball Institute and HHMI, New York University Langone Medical Center, New York City, NY 10016, USA.
³⁸ Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
³⁹ Cell Biology Program, The Hospital for Sick Children (SickKids), Toronto, ON M5G 0A4, Canada.
⁴⁰ Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
⁴¹ Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
⁴² CABD (Andalusian Centre for Developmental Biology), CSIC-UPO-JA, Seville 41013, Spain.
⁴³ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴⁴ Department of Neurobiology, Stanford University, Stanford, CA 94305, USA.
⁴⁵ Department of Biology, New York University, New York, New York 10003, USA.
⁴⁶ Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
⁴⁷ European Molecular Biology Laboratory, European Bioinformatics Institute, EMBL-EBI, Wellcome Trust Genome Campus, Hinxton CB10 1SD, UK.
⁴⁸ Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
⁴⁹ Department of Physiology, Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA.
⁵⁰ Howard Hughes Medical Institute, San Francisco, CA, USA.
⁵¹ The Hebrew University of Jerusalem, Jerusalem, Israel.
⁵² Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
⁵³ Department of Bioengineering and Biophysics Graduate Program, Stanford University, Stanford, CA 94305, USA.
⁵⁴ Université Côte d'Azur, CNRS, INSERM, iBV, France.
⁵⁵ Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA.
⁵⁶ University of California, Santa Barbara, CA 93106, USA.
⁵⁷ Uppsala University, Sweden.
⁵⁸ Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁵⁹ Institut Jacques Monod, Centre National de la Recherche Scientifique-UMR 7592, Université Paris Diderot, Paris, France.
⁶⁰ Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.
⁶¹ Donnelly Centre for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E1, Canada.
⁶² Skirball Institute, Department of Cell Biology and HHMI, New York University Langone Medical Center, New York City, NY 10016.
⁶³ Department of Biology, Syracuse University, Syracuse, NY, USA.
⁶⁴ Department of Evolutionary Biology, University of Vienna, Vienna, Austria.
⁶⁵ Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.
⁶⁶ European Bioinformatics Institute (EMBL/EBI), Wellcome Trust Genome Campus, Cambridge, UK.
⁶⁷ Department of Biology, New York University, New York, NY 10003, USA.
⁶⁸ Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3DY, UK.
⁶⁹ The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
⁷⁰ Skirball Institute, Faculty of Medicine, New York University, New York, NY 10016.
⁷¹ Genomics Core, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD, USA.
⁷² Graduate Program in Genetics and Genomics, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030 USA.
⁷³ Centre for Neural Circuits and Behaviour, University of Oxford, Tinsley Building, Mansfield Road, Oxford OX1 3TA, UK.
⁷⁴ Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA.
⁷⁵ Laboratory for Systems Biology of Neural Tissue Differentiation, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrueck Centre for Molecular Medicine (MDC) in the Helmholtz Association, Robert-Roessle-Strasse 12, 13125 Berlin, Germany.

^# Contributed equally.

PMID: 35239393
PMCID: PMC8944923
DOI: 10.1126/science.abk2432

Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly

Hongjie Li et al. Science. 2022.

. 2022 Mar 4;375(6584):eabk2432.

doi: 10.1126/science.abk2432. Epub 2022 Mar 4.

Authors

Affiliations

¹ Howard Hughes Medical Institute, Department of Biology, Stanford University, Stanford, CA 94305, USA.
² Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA.
³ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
⁴ VIB-KU Leuven Center for Brain and Disease Research, KU Leuven, Leuven 3000, Belgium.
⁵ Laboratory of Computational Biology, Department of Human Genetics, KU Leuven, Leuven 3000, Belgium.
⁶ Department of Bioengineering, Stanford University, Stanford, CA, USA.
⁷ Department of Applied Physics, Stanford University, Stanford, CA, USA.
⁸ Chan Zuckerberg Biohub, San Francisco CA, USA.
⁹ Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.
¹⁰ Swiss Institute of Bioinformatics, CH-1015 Lausanne, Switzerland.
¹¹ Bioinformatics Competence Center, EPFL, Switzerland.
¹² Department of Computer Science, Stanford University, Stanford, CA 94305, USA.
¹³ Department of Cell and Tissue Biology, University of California, San Francisco, CA 94143, USA.
¹⁴ Department of Life Science, College of Natural Science, Hanyang University, 04763 Seoul, Republic of Korea.
¹⁵ Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.
¹⁶ Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA.
¹⁷ Aix-Marseille University, CNRS, IBDM (UMR 7288), Turing Centre for Living Systems, 13009 Marseille, France.
¹⁸ Institute of Physiology and Pathophysiology, Department of Molecular Cell Physiology, Philipps-University, Marburg, Germany.
¹⁹ Department of Anatomy, University of California, San Francisco, CA 94143, USA.
²⁰ Behavior and Metabolism Laboratory, Champalimaud Research, Champalimaud Centre for the Unknown, Lisbon, Portugal.
²¹ National Center of Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
²² Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Kidney and Digestive Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
²³ Centre for Neural Circuits and Behaviour, University of Oxford, Oxford OX1 3SR, UK.
²⁴ Department of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
²⁵ Molecular Biosciences Division, Cardiff University, Cardiff CF10 3AX, UK.
²⁶ Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
²⁷ Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA 19104, USA.
²⁸ The Penn Institute for Regenerative Medicine, Philadelphia, PA 19104, USA.
²⁹ Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
³⁰ Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
³¹ Immunology Discovery, Genentech, Inc., South San Francisco, CA 94080, USA.
³² MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
³³ Department of Biology, Syracuse University, Syracuse, NY 13244 USA.
³⁴ Biomedical Sciences Department, Florida State University, Tallahassee, FL, USA.
³⁵ Department of Genetics, Harvard Medical School, Boston, MA, USA.
³⁶ Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
³⁷ Skirball Institute and HHMI, New York University Langone Medical Center, New York City, NY 10016, USA.
³⁸ Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037, USA.
³⁹ Cell Biology Program, The Hospital for Sick Children (SickKids), Toronto, ON M5G 0A4, Canada.
⁴⁰ Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
⁴¹ Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
⁴² CABD (Andalusian Centre for Developmental Biology), CSIC-UPO-JA, Seville 41013, Spain.
⁴³ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴⁴ Department of Neurobiology, Stanford University, Stanford, CA 94305, USA.
⁴⁵ Department of Biology, New York University, New York, New York 10003, USA.
⁴⁶ Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
⁴⁷ European Molecular Biology Laboratory, European Bioinformatics Institute, EMBL-EBI, Wellcome Trust Genome Campus, Hinxton CB10 1SD, UK.
⁴⁸ Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
⁴⁹ Department of Physiology, Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA, USA.
⁵⁰ Howard Hughes Medical Institute, San Francisco, CA, USA.
⁵¹ The Hebrew University of Jerusalem, Jerusalem, Israel.
⁵² Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
⁵³ Department of Bioengineering and Biophysics Graduate Program, Stanford University, Stanford, CA 94305, USA.
⁵⁴ Université Côte d'Azur, CNRS, INSERM, iBV, France.
⁵⁵ Biodesign Institute, Arizona State University, Tempe, AZ 85281, USA.
⁵⁶ University of California, Santa Barbara, CA 93106, USA.
⁵⁷ Uppsala University, Sweden.
⁵⁸ Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
⁵⁹ Institut Jacques Monod, Centre National de la Recherche Scientifique-UMR 7592, Université Paris Diderot, Paris, France.
⁶⁰ Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.
⁶¹ Donnelly Centre for Cellular and Biomolecular Research, Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E1, Canada.
⁶² Skirball Institute, Department of Cell Biology and HHMI, New York University Langone Medical Center, New York City, NY 10016.
⁶³ Department of Biology, Syracuse University, Syracuse, NY, USA.
⁶⁴ Department of Evolutionary Biology, University of Vienna, Vienna, Austria.
⁶⁵ Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.
⁶⁶ European Bioinformatics Institute (EMBL/EBI), Wellcome Trust Genome Campus, Cambridge, UK.
⁶⁷ Department of Biology, New York University, New York, NY 10003, USA.
⁶⁸ Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3DY, UK.
⁶⁹ The Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.
⁷⁰ Skirball Institute, Faculty of Medicine, New York University, New York, NY 10016.
⁷¹ Genomics Core, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, MD, USA.
⁷² Graduate Program in Genetics and Genomics, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030 USA.
⁷³ Centre for Neural Circuits and Behaviour, University of Oxford, Tinsley Building, Mansfield Road, Oxford OX1 3TA, UK.
⁷⁴ Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, TX 77030, USA.
⁷⁵ Laboratory for Systems Biology of Neural Tissue Differentiation, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrueck Centre for Molecular Medicine (MDC) in the Helmholtz Association, Robert-Roessle-Strasse 12, 13125 Berlin, Germany.

^# Contributed equally.

PMID: 35239393
PMCID: PMC8944923
DOI: 10.1126/science.abk2432

Abstract

For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

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Conflict of interest statement

Competing interests: H. Jasper, N.S. Katheder and X.T. Cai are employees of Genentech, Inc. Other authors declare no competing interests.

Figures

**Figure 1.. Overview of the Fly Cell Atlas**
**(A)** Experimental platform of snRNA-seq using 10x Genomics and Smart-seq2 (SS2). **(B)** Data analysis pipeline and data visualization using SCope (17) and ASAP (18). **(C)** Two versions of 10x datasets: *Relaxed* and *Stringent*. tSNE colors based on gene expression: *grh* (epithelia, red), *Mhc* (muscle, green) and *Syt1* (neuron, blue). Red arrow denotes an artefactual cluster with co-expression of all three markers in the *Relaxed* dataset. **(D)** tSNE visualization of cells from the *Stringent* 10x dataset and Smart-seq2 (SS2) cells. 10x cells are from individual tissues. Integrated data is colored by tissue (left) and platform (right). **(E)** Tissue-level comparison of the number of detected genes between 10x and Smart-seq2 platforms. **(F)** Number of cells for each tissue by 10x and Smart-seq2. Male and female cells are indicated. Mixed cells are from pilot experiments where flies were not sexed. Different batches are separated by vertical white lines. **(G)** All 10x cells from the *Stringent* dataset clustered together; cells are colored by tissue type. Tissue names and colors are indexed in F.

**Figure 2:. Cell type annotation for dissected tissues**
**(A)** Illustration of 15 individual tissues. 12 sequenced separately from males and females, 3 sex-specific. Fat body, oenocyte, and tracheal nuclei were labeled using a tissue-specific GAL4 driving UAS-nuclearGFP. **(B)** tSNE plot with annotations for body wall from the *Stringent* 10x dataset. *1, epidermal cells of the abdominal posterior compartment. *2, epidermal cells specialized in antimicrobial response. **(C)** UMAP plot with annotations for the testis from the *Relaxed* 10x dataset. **(D)** tSNE plots of the other 13 tissues from the *Stringent* 10x dataset. Detailed annotations are in fig. S6–S18. **(E)** Number of unique annotations for each tissue. Fractions of annotated cells over all analyzed cells from the *Relaxed* dataset are indicated in red.

**Figure 3:. Whole-head and whole-body sequencing leads to full coverage of the entire fly**
**(A)** tSNE of the whole-head sample with 81 annotated clusters. See fig. S22 for full cell types. Many cells in the middle (gray) are unannotated, most of which are central brain neurons. **(B)** tSNE of the whole-body sample with 33 annotated clusters, many of which can be further divided into sub-clusters. Cells in gray are unannotated. See fig. S23 for full cell types. **(C)** (left) tSNE of the entire dataset colored by standardized tissue enrichment, leading to the identification of head- and body-specific clusters. (right) Bar plots showing tissue composition (head, body, or dissected tissues) for different clusters at Leiden resolution 50. **(D)** Examples of head- and body-specific clusters. **(E)** Integration of a brain scRNA-seq dataset with the head snRNA-seq for label transfer. Outlined are example clusters revealed by the head snRNA-seq dataset but not by the brain scRNA-seq datasets, including epithelial cells (EPI), photoreceptors (PRs), olfactory receptor neurons (ORNs), and muscle cells (MUS). **(F)** Subclustering analysis reveals types of photoreceptors, including inner and outer photoreceptors, with the inner photoreceptors further splitting into R7 and R8 types, and mushroom body Kenyon cells comprising three distinct types: α/β, α’/β’ and γ.

**Figure 4:. Cross-tissue analyses of common cell classes**
**(A)** Overview of main cell classes identified throughout the fly cell atlas. Som. pre., somatic precursor cells; male repr. and fem. repr., male and female reproductive system; male germ. and fem. germ., male and female germline cells. **(B)** tSNE plots showing expression of four markers in four common cell classes. **(C)** Composition of whole head and body samples, showing a shift from neurons to epithelial and muscle cells. Composition of the entire fly cell atlas shows enrichment for rarer cell classes compared to the whole-body sample. **(D)** Cross-tissue analysis of hemocytes reveals different cell states of plasmatocytes. Annotations marked as blue are hemocytes containing markers of different cell types, including lymph gland posterior signaling center (LGP), muscle (MUS), antenna (ANT), neurons (NEU), photoreceptor (PR), male accessory glands (MAG), glia (G), male testis and spermatocyte (MS), olfactory-binding proteins (OBP), and heat-shock proteins (Hsp). Other abbreviations show top marker gene(s) in red. Plasmatocytes and crystal cells are indicated. On the right are genes showing compartmentalized expression patterns within the plasmatocyte cluster. **(E)** Cross-tissue analysis of muscle cells reveals subdivision of the visceral muscle cells based on neuropeptide receptors. Annotations marked as blue are muscle cells containing markers of different cell types, including neuron (NEU) and male testis and spermatocyte (MS). Muscle cells from three body parts are indicated: head muscle (HEAD), body muscle (BODY), and testis muscle (TESTIS). Other annotated muscle types include indirect flight muscle (IFM), ovarian sheath muscle (OSM), abdominal visceral muscle (ABD), *dpy* expressing muscle (DPY), visceral muscle of the midgut *AstC-R2* (VMM-A), visceral muscle of the crop *MsR1* (VMC-M), visceral muscle of the midgut *Dh31-R* (VMM-D), and visceral muscle *CCAP-R* (VM-C). *Pdfr* is expressed in all visceral muscle cells, including the ovarian sheath muscle; other four receptor genes (*AstC-R2, MsR1, Dh31-R, CCAP-R*) are expressed in different gut visceral muscle types.

**Figure 5:. Transcription factor (TF) pleiotropy versus cell-type specificity**
**(A)** Heatmap showing the expression of key marker genes and unique TF profiles for each of the annotated cell types. TFs were selected based on *tau* score. Cell types were grouped based on hierarchical terms: CNS neurons (N), sensory organ cells (S), epithelial cells (E), muscle cells (M), glia (G), fat cells (F), oenocytes (O), hemocytes (H), (fe)male reproductive system and germline (MR, MG, FR, FG), excretory system (X), tracheal cell (T), gland (L), cardiac cell (C), somatic precursor cell (P). **(B)** A network analysis of TFs and cell classes based on similarity of ontology terms, reveals unique and shared TFs across the individual tissues. **(C)** Heatmap showing the expression of unique TFs per cell class. Factors from the literature are highlighted. **(D)** *Glass* is uniquely expressed in photoreceptors and cone cells in the head. **(E)** Overview of the *Glass* regulon of 444 target genes, highlighting known photoreceptor marker genes. **(F)** Gene expression comparison across broad cell types. Only sets with more than 10 genes are shown. The left bar graph shows the number of uniquely expressed genes for each tissue. The top bar graph shows the gene age in branches, ranging from the common ancestor to *Drosophila melanogaster*-specific genes (http://gentree.ioz.ac.cn). See fig. S34 for tissue-based comparison.

**Figure 6.. Sex-biased expression and trajectory analysis of testis cell lineages**
**(A)** Simplified sex determination pathway. Sex chromosome karyotype (XX) activates Sex-lethal (Sxl) which regulates transformer (Tra), resulting in a female Dsx isoform (Dsx^F). In XY (or X0) flies, Sxl and Tra are inactive (light gray) and the male-specific Dsx^M is produced. **(B)** Top, Dsx expression and female- and male-biased expression projected onto tSNE plots of all female (left column) and male (right column) cells except reproductive tissue cells (Table S4 and S5). female- and male-biased expression measured as the percentage of genes in the cluster showing biased expression in favor of the respective sex (Table S6). These percentage values were computed for each annotated cluster and those cluster-level values were projected onto the individual cells in the corresponding clusters. For all four tSNE plots, values outside the scale in the heatmap key are represented by the closest extreme color (> and < signs in the scale). **(C)** Scatter plot of female- and male-bias values across non-reproductive cell clusters defined as % sex-biased genes (at least 2-fold change with FDR < 0.05 on Wilcoxon test and BH correction) in the cluster (Table S6). Data point size indicatess cell numbers per cluster (key). Selected clusters are labeled, with those from excretory cells highlighted (brown). MT, Malpighian tubule. **(D)** Box plots showing the relationship between *dsx* gene expression and sex-biased expression (Table S5). Clusters (B) were partitioned into the set of clusters with Dsx expression (*dsx*⁺) or not (no/low) using *dsx* expression in germ cells as an expression cut-off. Each box shows hinges at first and third quartiles and median in the middle. The upper whisker extends from the upper hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the inter-quartile range, or distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Outliers are not shown. p-values are based on Wilcoxon test. **(E–G)** Trajectory of testis subsets. We used slingshot to infer a possibly branching trajectory for spermatogonia-spermatocytes (E), spermatids (F), and early cyst cells (G). Shown are the trajectories on a UMAP (top) and the expression patterns of the strongest differentially expressed genes, together with the smoothed proportions of annotated cells and average number of unique molecular identifiers (UMIs) along the trajectory (bottom).

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References

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Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly

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Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly

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